Transcript
Page 1: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

Two-Photon Small Molecule Enzymatic ProbesLinghui Qian,† Lin Li,*,‡ and Shao Q. Yao*,†

†Department of Chemistry, National University of Singapore 117543, Singapore‡Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic InnovationCenter for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, P. R. China

CONSPECTUS: Enzymes are essential for life, especially inthe development of disease and on drug effects, but as wecannot yet directly observe the inside interactions and onlypartially observe biochemical outcomes, tools “translating”these processes into readable information are essential forbetter understanding of enzymes as well as for developingeffective tools to fight against diseases. Therefore, sensitivesmall molecule probes suitable for direct in vivo monitoring ofenzyme activities are ultimately desirable. For fulfilling thisdesire, two-photon small molecule enzymatic probes(TSMEPs) producing amplified fluorescent signals based onenzymatic conversion with better photophysical properties anddeeper penetration in intact tissues and whole animals havebeen developed and demonstrated to be powerful in addressing the issues described above. Nonetheless, currently availableTSMEPs only cover a small portion of enzymes despite the distinct advantages of two-photon fluorescence microscopy. In thisAccount, we would like to share design principles for TSMEPs as potential indicators of certain pathology-related biomarkerstogether with their applications in disease models to inspire more elegant work to be done in this area. Highlights will beaddressed on how to equip two-photon fluorescent probes with features amenable for direct assessment of enzyme activities incomplex pathological environments. We give three recent examples from our laboratory and collaborations in which TSMEPs areapplied to visualize the distribution and activity of enzymes at cellular and organism levels. The first example shows that we coulddistinguish endogenous phosphatase activity in different organelles; the second illustrates that TSMEP is suitable for specific andsensitive detection of a potential Parkinson’s disease marker (monoamine oxidase B) in a variety of biological systems from cellsto patient samples, and the third identifies that TSMEPs can be applied to other enzyme families (proteases). Indeed, TSMEPshave helped to uncover new biological roles and functions of a series of enzymes; therefore, we hope to encourage more TSMEPsto be developed for diverse enzymes. Meanwhile, improvements in the TSMEP properties (such as new two-photonfluorophores with longer excitation and emission wavelengths and strategies allowing high specificity) are also indispensable forproducing high-fidelity information inside biological systems. We are enthusiastic however that, with these efforts and widerapplications of TSMEPs in both research studies and further clinical diagnoses, comprehensive knowledge of enzymecontributions to various physiologies will be obtained.

■ INTRODUCTION

Chemical probes are dedicated to illustrating biologicalprocesses as well as providing practical tools for modulatingthem at the interface of advanced chemistry and biology.1

Techniques developed in this field include making theinformation inside the biological system “readable” with probesand enabling artificial regulation of physiological functions withnewly synthesized/discovered drug candidates, which wouldundisputedly promote development in disease diagnosis,therapy, and prevention.2 Within living organisms, proteinsact as front-line workers that participate in virtually everyprocess to maintain homeostasis. Over the years, our group hasutilized various platforms to facilitate the study of proteins andtheir molecular interactions.3−9 Specifically, we are highlyinterested in enzymes due to their intimate association withdiseases10 and druggability.11,12 Comprehensive illustration ofenzyme functions, together with approaches for precise

detection of their activities under both healthy and pathologicalconditions, is fundamental for disease diagnosis and treatment.For the precise evaluation of enzyme activities that are

functionally regulated by a series of post-translationalmechanisms in a dynamic manner, fluorescence imaging standsout for its ability to sensitively visualize biological targets andprocesses with high temporal-spatial resolution, and dramaticprogress has been made over the last several decades.13−17 Adiverse array of small molecule-based fluorescent probes withdesired photophysical properties and advantageous cellpermeability or even organelle specificity, binding affinity,kinetics, and chemical tractability are available nowadays.18

Among them, enzymatic probes (also known as substrate-basedprobes)17,19 are competitive for bioimaging because of two

Received: November 23, 2015Published: April 5, 2016

Article

pubs.acs.org/accounts

© 2016 American Chemical Society 626 DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

Page 2: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

imponderable advantages. One, they can mimic the intrinsicmetabolic process and produce sufficient signals to bedistinguished among complicated biological components, andthe other, as signals originate exclusively from reactionscatalyzed by the defined enzyme under native conditions, it isa direct measure of enzyme activity rather than proteinexpression level. It should be noted that, in most cases, whatmatters most in vivo is the enzyme at work, especially in thepresence of endogenous inhibitors, such that the amount ofprotein may remain unchanged while the activity has alreadychanged.17

Although an increasing number of probes have beendeveloped to investigate various enzymes, available probes arelimited for in-cell fluorescence imaging and cannot be appliedto the majority of disease models constructed with tissues andanimals. Fluorophores utilizing a longer wavelength forexcitation can achieve deeper penetration due to reducedphoton scattering (photon scattering scales as λ−α, where λ isthe wavelength and α = 0.2−4 for biological tissues),20 and thewavelength range of 650−1450 nm falls in the region of thespectrum with the lowest absorption in tissue (absorbance bywater, melanin, proteins, and hemoglobin are high between 200and 650 nm) and has minimal autofluorescence.21 Remarkably,near-infrared (NIR) fluorophores activatable by one-photonhave attracted significant attention in bioimaging due toinexpensive diode laser excitation. However, there are relativelyfew classes of NIR fluorophores readily available, including thephthalocyanines, cyanine, and squaraine dyes.22 With con-straints in the large conjugated structure, NIR fluorophoresoften suffer from aqueous insolubility, insufficient stability inbiological systems, and low quantum yields, leaving subsequentfunctionalization or optimization of NIR fluorophores challeng-ing.23,24 Alternatively, for multiphoton dyes absorbing two ormore photons simultaneously for each transition event,25,26 anNIR laser is used to excite the fluorophore, enabling deep-tissuepenetration while at the same time allowing more flexibility inthe structures.27 Moreover, because the signal (S) dependssupralinearly (S ∝ In, n = 2 for two-photon dyes) on the densityof photons (or on the excitation intensity, I), the fluorescence isconfined to the vicinity of the focal point (∼1 μm3).27,28 Thislocalized excitation not only provides intrinsic three-dimen-sional resolution in fluorescence microscopy but also lowersphotodamage and photobleaching. Very recently, three-photonfluorescence microscopy has been shown to image up to 3.5mm deep into tissue.20 This is undoubtedly a significant step

toward enlarging the depth for imaging, but collective effortsare still needed for wide applications. In particular, thedevelopment of two-photon fluorescence microscopy(TPFM) has been propelled by rapid technological advancesin laser scanning microscopy, fluorescent probe synthesis, andcomputational three-dimensional image reconstruction.26 Fur-thermore, increasing knowledge in chemistry and photophysicsalso enables the development of a TP fluorophore with betterphotophysical properties.29

A series of TP probes targeting biothiols, reactive oxygenspecies (ROS), and metal ions have already been establishedwith outstanding advantages over one-photon (OP)probes.28,30,31 However, TP probes for enzymes that areindispensable in homeostasis maintenance remain under-developed. For TPFM to be widely adopted for bioimagingof enzyme activities and to address some controversialphenomena in biology, it is essential to produce enzymaticprobes that not only incorporate a TP fluorophore but alsohave improved properties to address the void, such as betterselectivity/sensitivity and higher spatiotemporal resolution in anoninvasive manner. To highlight the “target-guided” design intwo-photon small molecule enzymatic probes (TSMEPs), inthis Account we will divide available TSMEPs according totheir targets (1, endogenous phosphatase activity in differentorganelles; 2, the potential Parkinson’s disease marker mono-amine oxidase B; and 3, proteases) to introduce principles usedfor developing practical TSMEPs as well as inspirations forbiological issues.

■ PRINCIPLES FOR TSMEP DESIGN

By definition, TSMEPs can be recognized by an enzyme in situand generate amplified signals through intrinsic multiturnoverenzymatic reactions.17,19,32 Strategies employing this enzymaticconversion to “switch-on” the probe (changes in wavelengthand/or intensity) usually apply one of the followingmechanisms: (1) Forster resonance energy transfer (FRET)(Figure 1a),33−36 (2) intramolecular charge transfer (ICT)(Figure 1b),37−39 and (3) transformation of a latent fluorescentmolecule into a fluorescent one upon conjugation reconstruc-tion (Figure 1c).40 In probes relying on FRET, a fluorophoreand quencher pair are attached to each other within a certaindistance via an enzyme-cleavable linker to generate aprequenched probe that restores fluorescence upon release ofthe quencher by a specific enzyme (Figure 1a). The ICTapproach often asks for a change in electron-donating and/or

Figure 1. Representative strategies involved in two-photon small molecule enzymatic probes. (a) Enzyme-activatable probes based on FRET. (b)Substrate-like fluorogenic probes based on intramolecular charge transfer. (c) Reaction-based reconstruction of the conjugating system in themolecule leading to a fluorescence change.

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

627

Page 3: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

-withdrawing groups within the molecule by enzymaticconversion (Figure 1b). A more specific method is themodulation of a molecular conjugation system based onenzyme-mediated reactions (Figure 1c), leading to a photo-physical property change of the probe. Of note, photon-induced electron transfer (PeT) is another powerful method tointroduce a change in fluorescence. For probes constituting adonor and an acceptor, certain chemical reactions eliminate thecharge transfer between them under excitation, which restoresthe fluorescence. A wealth of TP probe-detecting metal ions aredecorated with PeT for excellent “turn-on” efficiency.31

Although only partially involved in a few enzymatic probes,37

PeT is not included as a general mode here. Overall, a desirableTSMEP should undergo a detectable fluorescence change afterthe chemical modification catalyzed by the target enzyme.28

The following requirements should also be taken intoconsideration during probe design: (1) biocompatibility (watersolubility, cell permeability, cytotoxicity, and chemical stabilityunder native conditions), (2) sufficient in vitro potency andselectivity to indicate and fulfill its cellular or in vivoperformance, (3) sensitivity, especially for proteins at lowabundance, and (4) easy preparation (feasible synthetic routeor readily available source). However, we can generally onlybuild valid and objective rule-based parameters for biologicalsystems according to those existing quality probes. Whenapplied under physiological conditions, the quality probediscovers its own path to validation.41

■ TSMEPS FOR PHOSPHATASES

Phosphatases are a large family of enzymes that participate insignaling networks by removing the phosphate group from theirsubstrates. Of the various types of phosphatases, proteintyrosine phosphatases (PTPs), which mainly catalyze thedephosphorylation of pTyr residues in proteins, are implicatedin many human diseases once under defective regulation.42,43

There are over 100 members in the PTP family that differ intheir protein structure, substrate specificity, and subcellularlocalization.42

Consequently, biological and chemical approaches capable ofreporting phosphatase activities would provide invaluableinsights into how these enzymes work under physiologicalsettings and whether they are indicative during diseasedevelopment. In 1939 as pioneering work in enzymatic assays,Gomori developed a method to visualize phosphatase activity intissue sections based on the idea that a phosphate released fromglycerophosphate via enzyme catalysis could be detected interms of the formation of an insoluble salt with Ca2+.43

Thereafter, numerous strategies aimed at identifying phospha-tase activities in cells were established.44,45 However, to makethe detection amenable to a wider range of biologicalspecimens, several issues remained to be addressed, includinghigher sensitivity, better spatial resolution, and improveddetection depth in live cells and tissues.With these goals in mind, we came up with the first TSMEP

for PTPs, TP-PTP, which allows signal retention andamplification without inactivation of the target enzyme forhelping quinone methide chemistry.33 The quinone methideintermediate has a distinct property that, once formed, it willdiffuse from the active site at a rate competitive with thesubsequent labeling process, thus causing labeling to occuraway from the catalytic active site of the target enzyme.46 Bytaking advantage of the reactive yet diffusible nature of thisintermediate, both visualization of enzyme localization andfluorescent signal amplification to sensitively report endoge-nous multiturnover enzyme activity can be achieved. With amandelic acid core, three modular components, including anenzyme substrate warhead (WH), a fluorescence reporter (F),and a quencher (Q), were strategically built around it (Figure2a).33 For targeting PTPs as well as to enable temporal controlof imaging in live cells, a phosphate group “caged” withphotolabile groups (2-nitrobenzyloxy) was incorporated. Mean-while, a FRET pair of dabcyl quenchers and the well-known TPfluorophore, 2-hydroxy-4,6-bis(4-(diethylamino)styryl)-pyrimidine, were attached to the mandelic acid core andproximal to each other, offering a substantial quenching effect(Figure 2b). Once such low-background probes entered cells,UV irradiation can remove the caging group to present the

Figure 2. First TSMEP for protein tyrosine phosphatases (PTPs). (a) Overall design principle for the enzymatic probe with an anchoring propertyafter activation. (b) Structure of TP-PTP in this study. (c) Two-photon images (λex = 800 nm, taken on a Leica TCS SP5X confocal microscopesystem) of endogenous PTPs in HeLa cells using TP-PTP with (left) and without (right) UV exposure.33

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

628

Page 4: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

naked phosphate group under control. In the presence of PTPs,the quencher was removed by 1,6-elimination to liberatedistinct fluorescence (“turn-on”) as well as to covalently label

neighboring nucleophiles of the enzyme active site to indicatethe localization of enzymes. With such a probe in hand, TPFMof HeLa cells was carried out and indicated strong PTP

Figure 3. Organelle-specific TSMEPs for phosphatases. (a) (top) Schematic of our CPP-conjugated two-photon fluorogenic probes; (bottom) TPimaging (λex = 800 nm) of endogenous phosphatase activities in live HeLa cells demonstrating targeted localizations with designed CPP with YP1 in1−4, YP2 in 5−8, and YP3 in 9−12. (b) Structures of reporters (Y1−4) derived from the well-known TP fluorophore (Y1*) in this study. (c)Images of endogenous phosphatase activities in fresh brains of Drosophila using Y2 and uncaged YP2 after incubation with the probes (20 μM) for 6h under TPFM (λex = 800 nm) at a depth of ∼110 μm. (1−3) Y2, insets are images obtained from the brain treated with tyrosine phosphataseinhibitor Na3VO5. (4−6) Uncaged YP2. (3, 6) Enlarged images of the red boxes in (2) and (5) with 40× magnification.39

Figure 4. (a) Overall strategy of our switchable TP membrane tracer pair, Flu7/Q12 (structures shown at right), which is capable of imagingmembrane-associated receptor-like protein tyrosine phosphatase (RPTP) activities. (b) (top) One-photon images of HepG2 cells treated with Flu7(1 equiv), Q12 (2 equiv), and UV irradiation successively; (bottom) total fluorescence intensities of the images (green) compared with those fromthe isolated membrane fraction of the same treated cells (blue). (c) Effect of anions (10 equiv) on Flu7/Q12 fluorescence. (d) Membrane-associatedRPTP activity of eight different mammalian cell lines measured by FACS (green) and membrane fractionation (blue) experiments. (inset)Correlation between these two experiments, fitted by linear regression, giving R2 = 0.98. (e) TPFM (λex = 770 nm) of RPTP activities in fresh brainsof Drosophila using Flu7/uncaged Q12 at a depth of ∼110 μm: (1) bright field image of brain, (2) Drosophila brain treated with Flu7 (20 μM) for 2h, (3) further incubation with uncaged Q12 (100 μM) for 20 min, and (4) after further incubation for 6 h. (bottom) Magnification (4×) of theimages in the red boxes in panels 2−4.35

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

629

Page 5: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

activities in cytosol (Figure 2c). Meanwhile, additional tags,such as biotin for further protein profiling studies or cell-penetrating peptides (CPPs) to facilitate cellular entrance, canbe introduced when needed. Such an activatable TSMEP wasintroduced for the first time in the detection of enzymeactivities; more significantly, the modular design here can beapplied to other hydrolytic enzymes (e.g., all four major classesof proteases) as well.One of the driving issues in the development of potent small

molecule probes for high-resolution imaging is their very fastdiffusion. In addition to molecules described above using ahighly reactive quinone methide intermediate to “fix” thefluorescent adduct near the site of the reaction, a “targeting”moiety is another choice. It can be conjugated to the probe forspecific delivery and enhanced subcellular retention with lessinterference to protein activities than probes containing reactivegroups. To this end, fluorogenic probes employing ICT andfurnished with targeting moieties were developed (Figure 3a).39

Substitution of 4-diethylamino in Y1* to 4-hydroxy gave Y1with a TP action cross-section (δ·Φ = 82 GM) comparable tothose of other common TP fluorophores (Figure 3b).47 Onceattached by the electron-withdrawing phosphate group(phosphatase-responsive warhead) at two phenolic groups(Y2), a significantly reduced conjugated π-electron system ledto much weaker red-shifted fluorescence emission of the probe

as the “OFF” state. For sufficient delivery of the probes withoutpremature interaction with phosphatases outside the desiredorganelle (phosphatase activity was ubiquitously present atmost intracellular spaces and organelles), the phosphate groupswere protected by photolabile moieties (i.e., 2-nitrobenzylgroup in Y3/Y4). Moreover, the targeting groups terminatedwith an azide can easily be assembled to the alkyne derivativeY4 for detection of localized enzyme activities. Fourrepresentative CPPs specific for endoplasmic reticulum (ER,linker-KKKRKV), plasma membrane (linker-KK (Palmitoyl)),mitochondria (linker-FxrFxK), and cancer cells (Ueeeeeeee-GGPLGLAG-rrrrrrrrr-linker; cleavable by matrix metallopro-teases that are overexpressed on the surface of numerous cancercells, including HeLa cells) were conjugated to give YP1−4,respectively (Figure 3a). After the probes accumulated at thetargeted location, the protecting group was removed by quickUV irradiation to interact with PTPs therein. Encouragingly,the “turn-on” fluorescence signal linked by CPPs can bespatially stable afterward, which benefits imaging (Figure 3a,bottom). As expected, this novel TP system succeeded inimaging endogenous phosphatase activities in Drosophila brainswith a detection depth over 100 μm (Figure 3c).One potential drawback of the former design is that the

localized signal may not all come from PTPs within thetargeted organelle, as the premature fluorophore “turned-on”

Figure 5. (a) Structure of the first TSMEP for MAOs and its “turn-on” mechanism.40 (b) Overall strategy using U1 for MAO-B-specific detection inlive mammalian cells, Drosophila, mice, and B lymphocytes isolated from PD patients. (c) Docked structures of CL (clorgyline, MAO-A-specificinhibitor) with MAO-A (left), PA (pargyline, MAO-B-specific inhibitor) with MAO-B (middle), and U1 with MAO-B (right). (d) (left) Relativefluorescence reading of U1 (2.0 μM) + brain lysates of 1, 6, and 12 month old (1/6/12-MO) mice with and without PA (green/purple). (inset) WBresults. (middle) TPFM (λex = 780) of the SNpc region of fresh 1 and 12 month mouse brain slices with (1)/(3) U1 (100 μM) and (2)/(4) U1(100 μM) + PA (200 μM) at 120 μm depth with 40× magnification. (right) Average fluorescence intensity profiles of mouse brain image panels (1−4). (e) U1-assisted detection of MAO-B for potential clinical diagnostics of PD in humans. (top) Relative-fluorescence reading of U1 (2.0 μM) inproteome lysates of human B lymphocytes obtained from PD patients (P4−6) and aged-matched normal controls (Ctrl4−6). (inset) WB results ofthe corresponding samples. (bottom) Relative-fluorescence reading of U1 (2.0 μM) in lysates prepared from fibroblasts of PD patients (P1−3) andthe corresponding age-matched controls (Ctrl). (inset) WB results of the corresponding samples. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3;Student’s t test, two-tailed in (d) and (e). (f) (left) TPFM (λex = 780 nm) of fresh brain slices of 20 day old parkin-null Drosophila with (1) U1 (100μM) and (2) U1 (100 μM) + PA (200 μM) at a depth of 100 μm with 40× magnification. (3) Reconstructed 3D TP image of (1). (right) Averagefluorescence intensity profiles of (1)/(2) at the indicated position.37

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

630

Page 6: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

by PTPs from somewhere else can also be driven to theorganelle by the CPPs. For addressing this, a novel “ON/OFF/ON” system was proposed to ensure the exclusive detection ofmembrane-associated, receptor-like protein tyrosine phospha-tases (RPTPs) (Figure 4a).35 First, Flu7, which contains the π-conjugated fluorene moiety modified with two 6-carbonaliphatic chains for membrane anchoring, could serve as a TPmembrane tracer as the “ON” state. The other two hydro-phobic tails in Flu7 were specifically capped with positivelycharged quaternary ammonium groups. Therefore, a negativelycharged pairing partner, Q12, consisting of a fluorescencequencher (Disperse Red 1) and a “photo-caged” phosphategroup, can bind to Flu7 through electrostatic interactions, thuseffectively quenching the fluorescence through intermolecularFRET (“OFF” state). Subsequently, upon UV irradiation, thedeprotected phosphate can undergo enzymatic dephosphor-ylation by membrane-associated RPTPs. Finally, the dephos-phorylated Q12 would dissociate from Flu7 to restore itsfluorescence (“ON” state). In this way, the recovered signalonly came from the membrane-associated RPTPs, and it wasfurther confirmed by a membrane fraction assay (Figure 4b).Interestingly, common biological anions showed little effect onFlu7/Q12 fluorescence (Figure 4c), indicating the associationof Flu7/Q12 was driven not only by electrostatic interactionsbut also by other noncovalent interactions like π−π stackingand hydrophobic interactions. For compatible live-cell imagingand strong restriction to membrane-associated PTPs, elevatedactivities of RPTP in cancer cell lines were differentiated by thisprobe from the normal cells (Figure 4d), consistent withreports that increased endogenous RPTP activity is responsiveto tumorigenesis in numerous cells and tissues.42 Ultimately,the utility of this system in TPFM was established successfullywhen imaging RPTP activity deep inside live Drosophila brains(Figure 4e). Similarly, the FRET strategy was also successfullyapplied by Xing et al. for real-time imaging of a cell surface-associated proteolytic enzyme (i.e., furin-like convertase)activities, where the fluorophore and quencher were linked bya cleavable peptide.48

■ TSMEP FOR MONOAMINE OXIDASE B

Monoamine oxidases (MAOs) are integral proteins of the outermitochondrial membrane that catalyze the oxidative deami-nation of monoamine neurotransmitters, including noradrena-line, adrenaline, and dopamine, along with the release ofhydrogen peroxide (H2O2).

49,50 With the ability to terminatephysiological actions of these neurotransmitters, the expression

level and enzymatic activities of MAOs are crucial to thenervous system. Two isoforms of this enzyme (MAO-A/-B)have been identified in humans, which differ in distribution,substrate selectivity, and inhibitor sensitivity, thus havingdistinct clinical significances.51,52 One example is the age-related neurodegenerative disorder Parkinson’s disease (PD),such that the expression of MAO-B but not MAO-A increaseswith age, and its activity is significantly enhanced in the brainsof PD patients.53 At present, there is no convenient diagnosticbiomarker for PD, and in the clinic, PD is mainly diagnosedbased on symptoms and neuroimaging tests, such as dopamine-based positron emission-computed tomography (PET) imagingwith exclusively high cost. With evidence suggesting MAO-B tobe a potential PD marker but not conclusive yet, it is urgent tolook for more practical means in MAO-B detection to explorethe unknowns and facilitate clinical diagnosis of PD.The majority of known fluorescence sensing systems for

MAOs are based on one-photon fluorophores;54−57 only Ahnand co-workers developed compounds 1a and 1b as TP probesfor MAOs, for the first time, by applying the known reaction-based sensing scheme (Figure 5a).40 A few of them are selectivefor MAO-A/-B,57 but they have not been well-investigated incentral neuron disease models or clinic samples. To fill up thisgap, U1, a TSMEP suitable for specific and sensitive detectionof MAO-B activities in a variety of biological systems includingestablished PD models, was developed recently (Figure 5b).37

U1 employed 2-methylamino-6-acetylnaphthalene (acedan,Flu1) as the TP reporter and 3-aminopropyl carbamate asthe MAO responsive unit. Compared with MAO-A, the activesite of MAO-B is longer and narrower and consists of an“entrance” cavity (290 Å3) and a hydrophobic “substrate” cavity(490 Å3) occupied by the redox-active isoalloxazine ring ofFAD coenzyme at the distal end; thus, U1 can fit into the“substrate” cavity of MAO-B snugly for distinct specificity(Figure 5c). Initially, the electron-poor carbamate bond in U1quenched the intrinsic fluorescence of acedan through a PeTand ICT mix effect. Upon MAO-B-catalyzed oxidation of theamino group to aldehyde, a propionaldehyde moiety is releasedspontaneously by β-elimination followed by self-immolativedischarge of CO2 to liberate the highly fluorescent Flu1. Withsuch outstanding contrast between background and signal, U1was indeed a probe suitable for continuous monitoring ofMAO-B activity in both live cells and tissues by TPFM. Asshown in Figure 5d and f, U1 can unambiguously report thedifference of MAO-B activities in PD models from the controlones, including age-associated increases in MAO-B expression

Figure 6. Overall strategy of the small molecule microarray (SMM)-guided, high-throughput discovery of cell permeable TSMEPs (ZK-1 and ZK-2)for imaging of endogenous cathepsin L.36

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

631

Page 7: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

(Figure 5d) and parkin-related regulation of MAO-B (Figure5f). Intriguingly, elevated MAO-B activities were detected inhuman B lymphocytes from PD patients by U1, suggesting thatthe MAO-B level in peripheral blood cells is a potentialbiomarker for rapid diagnosis of PD (Figure 5e). Given thehigh MAO-B selectivity provided by acedan, which also worksas a built-in fluorophore, a dual-purpose activity-based probecapable of in situ profiling and live-cell imaging of MAO-B wasdeveloped based on the key features of both U1 and theproteome profiling-enabled probes as the first example toaccomplish what all previously reported MAO-B imagingprobes failed to do.58

■ TSMEPS FOR PROTEASES

Among various enzymes, proteases are particularly attractivenot only because they are large in number but also due to theirroles in controlling numerous biological processes as signalingmolecules.59 According to their intrinsic property of proteolysis,probes aimed at them often contain a peptide derived fromtheir endogenous substrates for recognition. However, overlapin substrates between different proteases, especially those in thesame family, impedes their applications as precise sensors.Efforts have been made to improve selectivity among certainprotease subfamilies through screening for the optimal peptidesequence.60 For achieving this, high-throughput screening(HTS) platforms are desirable. Our group demonstrated aconvenient system by using small molecule microarray (SMM)as an HTS platform for the successful discovery of two potentinhibitors of cathepsin L, which were converted strategically tothe corresponding TSMEPs (Figure 6).36

■ CONCLUSIONS

Two-photon probes are indeed welcome in preclinicinvestigations and are promising for in vivo imaging withadvanced technologies, such as microendoscopy,61,62 asinformation closer to real physical conditions can be obtainedwith direct operation on tissue samples or whole animals.Therefore, the TSMEPs described above should be applied toexplore information that may be overlooked with traditionalmethods. Meanwhile, it is essential to enlarge the inventory ofTSMEPs to cover more medically interesting enzymes (mainlyhydrolases, oxidoreductases, and transferases as the mostcommon enzymatic drug targets)11 because TSMEPs availablenow only correspond to less than a dozen enzymes (i.e.,phosphatases, monoamine oxidase, and proteases). For this tobe accomplished, TP fluorophores with better photophysicalproperties (e.g., brighter and longer excitation and emissionwavelengths, superior stability) that are amenable to multicolorimaging are highly desirable. Meanwhile, long-standing issues inenzyme detection, such as sensitivity, selectivity, and bio-compatibility (e.g., permeability, less invasive for biologicalapplications) should be considered in novel TSMEP design.Attention should also be given to moving away from thedescription of an individual protein, instead viewing variousinteractions as a whole to understand the signaling network.Therefore, real-time as well as multicolor imaging allowingsimultaneous detection of several proteins will be helpful.Furthermore, quantification is becoming more appreciated influorescence imaging of enzymes to distinguish between normaland disease state; thereby, TSMEPs with internal inference arevery promising. We are enthusiastic that, with more effortdelivered to this field, next-generation TSMEPs possessing

greater capabilities will soon be developed to meet thesespecific needs.

■ AUTHOR INFORMATIONCorresponding Authors

*E-mail: [email protected].*E-mail: [email protected]

Grants from the Singapore National Medical Research Council(CBRG/0038/2013), the Ministry of Education (MOE2013-T2-1-048), Fundamental Studies of Perovskite Solar Cells(2015CB932200), National Natural Science Foundation ofChina (61505076), and Natural Science Foundation of JiangsuProvince (BK20140951) are acknowledged.Notes

The authors declare no competing financial interest.

Biographies

Linghui Qian received a B.S. in Pharmacy from Zhejiang University in2012 and is currently a Ph.D. student of Chemistry at the NationalUniversity of Singapore (NUS).

Lin Li received his B.S. and Ph.D. in chemistry from Anhui University.He is a Professor at Nanjing Tech University after postdoctoraltraining in Prof. Yao’s lab from 2009 to 2014 at NUS.

Shao Q. Yao graduated from Ohio State University with a B.S. inchemistry and received his Ph.D. from Purdue University. After hispostdoctoral research at UC Berkeley and Scripps Research Institute,he joined NUS in 2001 and is a Professor of Chemistry.

■ REFERENCES(1) Wijdeven, R. H.; Neefjes, J.; Ovaa, H. How Chemistry SupportsCell Biology: the Chemical Toolbox at Your Service. Trends Cell Biol.2014, 24, 751−760.(2) Pope, A. J. The Role of Chemical Biology in Drug Discovery.Chemical Biology: Approaches to Drug Discovery and Development toTargeting Disease, 1st ed.; Civjan, N., Ed.; John Wiley & Sons, Inc,2012; pp 1−21.(3) Chen, G. Y. J.; Uttamchandani, M.; Lue, R. Y. P.; Lesaicherre, M.-L.; Yao, S. Q. Array-Based Technologies and Their Applications inProteomics. Curr. Top. Med. Chem. 2003, 3, 705−724.(4) Uttamchandani, M.; Huang, X.; Chen, G. Y. J.; Tan, L.-P.; Yao, S.Q. Application of Microarrays in High-Throughput EnzymaticProfiling. Mol. Biotechnol. 2004, 28, 227−239.(5) Uttamchandani, M.; Li, J.; Sun, H.; Yao, S. Q. Activity-BasedProtein Profiling: New Developments and Directions in FunctionalProteomics. ChemBioChem 2008, 9, 667−675.(6) Uttamchandani, M.; Lu, C. H.; Yao, S. Q. Next GenerationChemical Proteomic Tools for Rapid Enzyme Profiling. Acc. Chem. Res.2009, 42, 1183−1192.(7) Kalesh, K. A.; Shi, H.; Ge, J.; Yao, S. Q. The Use of ClickChemistry in The Emerging Field of Catalomics. Org. Biomol. Chem.2010, 8, 1749−1762.(8) Su, Y.; Ge, J.; Zhu, B.; Zheng, Y.-G.; Zhu, Q.; Yao, S. Q. TargetIdentification of Biologically Active Small Molecules via in SituMethods. Curr. Opin. Chem. Biol. 2013, 17, 768−775.(9) Sun, H.; Chen, G. Y. J.; Yao, S. Q. Recent Advances in MicroarrayTechnologies for Proteomics. Chem. Biol. 2013, 20, 685−699.(10) Hanash, S. Disease Proteomics. Nature 2003, 422, 226−232.(11) Rask-Andersen, M.; Almen, M. S.; Schioth, H. B. Trends in theExploitation of Novel Drug Targets. Nat. Rev. Drug Discovery 2011, 10,579−590.(12) Copeland, R. A.; Harpel, M. R.; Tummino, P. J. TargetingEnzyme Inhibitors in Drug Discovery. Expert Opin. Ther. Targets 2007,11, 967−978.

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

632

Page 8: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

(13) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. CreatingNew Fluorescent Probes for Cell Biology. Nat. Rev. Mol. Cell Biol.2002, 3, 906−918.(14) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Fluorescence Imagingin Vivo: Recent Advances. Curr. Opin. Biotechnol. 2007, 18, 17−25.(15) Ueno, T.; Nagano, T. Fluorescent Probes for Sensing andImaging. Nat. Methods 2011, 8, 642−645.(16) Fernandez-Suarez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9,929−943.(17) Komatsu, T.; Urano, Y. Evaluation of Enzymatic Activities inLiving Systems with Small-molecular Fluorescent Substrate Probes.Anal. Sci. 2015, 31, 257−265.(18) Drake, C. R.; Miller, D. C.; Jones, E. F. Activatable OpticalProbes for the Detection of Enzymes. Curr. Org. Synth. 2011, 8, 498−520.(19) Edgington, L. E.; Verdoes, M.; Bogyo, M. Functional Imaging ofProteases: Recent Advances in The Design and Application ofSubstrate-Based and Activity-Based Probes. Curr. Opin. Chem. Biol.2011, 15, 798−805.(20) Hong, G.; Diao, S.; Chang, J.; Antaris, A. L.; Chen, C.; Zhang,B.; Zhao, S.; Atochin, D. N.; Huang, P. L.; Andreasson, K. I.; Kuo, C.J.; Dai, H. Through-Skull Fluorescence Imaging of the Brain in a NewNear-Infrared Window. Nat. Photonics 2014, 8, 723−730.(21) Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K.Review of Long-Wavelength Optical and NIR Imaging Materials:Contrast Agents, Fluorophores and Multifunctional Nano Carriers.Chem. Mater. 2012, 24, 812−827.(22) Frangioni, J. In Vivo Near-Infrared Fluorescence Imaging. Curr.Opin. Chem. Biol. 2003, 7, 626−634.(23) Escobedo, J. O.; Rusin, O.; Lim, S.; Strongin, R. M. NIR Dyesfor Bioimaging Applications. Curr. Opin. Chem. Biol. 2010, 14, 64−70.(24) Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. A Review of NIRDyes in Cancer Targeting and Imaging. Biomaterials 2011, 32, 7127−7138.(25) Denk, W.; Strickler, J. H.; Webb, W. W. Two-Photon LaserScanning Fluorescence Microscopy. Science 1990, 248, 73−76.(26) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W.Multiphoton Fluorescence Excitation: New Spectral Windows forBiological Nonlinear Microscopy. Proc. Natl. Acad. Sci. U. S. A. 1996,93, 10763−10768.(27) Helmchen, F.; Denk, W. Deep Tissue Two-Photon Microscopy.Nat. Methods 2005, 2, 932−940.(28) Kim, D.; Ryu, H. G.; Ahn, K. H. Recent Development of Two-Photon Fluorescent Probes for Bioimaging. Org. Biomol. Chem. 2014,12, 4550−4566.(29) Kim, D.; Moon, H.; Baik, S. H.; Singha, S.; Jun, Y. W.; Wang, T.;Kim, K. H.; Park, B. S.; Jung, J.; Mook-Jung, I.; Ahn, K. H. Two-Photon Absorbing Dyes with Minimal Autofluorescence in TissueImaging: Application to in Vivo Imaging of Amyloid-beta Plaques witha Negligible Background Signal. J. Am. Chem. Soc. 2015, 137, 6781−6789.(30) Yao, S.; Belfield, K. D. Two-Photon Fluorescent Probes forBioimaging. Eur. J. Org. Chem. 2012, 2012, 3199−3217.(31) Kim, H. M.; Cho, B. R. Small-Molecule Two-Photon Probes forBioimaging Applications. Chem. Rev. 2015, 115, 5014−5055.(32) Dean, K. M.; Palmer, A. E. Advances in Fluorescence LabelingStrategies for Dynamic Cellular Imaging. Nat. Chem. Biol. 2014, 10,512−523.(33) Hu, M.; Li, L.; Wu, H.; Su, Y.; Yang, P.-Y.; Uttamchandani, M.;Xu, Q.-H.; Yao, S. Q. Multicolor, One- and Two-Photon Imaging ofEnzymatic Activities in Live Cells with Fluorescently QuenchedActivity-Based Probes (qABPs). J. Am. Chem. Soc. 2011, 133, 12009−12020.(34) Zhang, C.-J.; Li, L.; Chen, G. Y. J.; Xu, Q.-H.; Yao, S. Q. One-and Two-Photon Live Cell Imaging Using a Mutant SNAP-TagProtein and Its FRET Substrate Pairs. Org. Lett. 2011, 13, 4160−4163.(35) Li, L.; Shen, X.; Xu, Q.-H.; Yao, S. Q. A Switchable Two-PhotonMembrane Tracer Capable of Imaging Membrane-Associated Protein

Tyrosine Phosphatase Activities. Angew. Chem., Int. Ed. 2013, 52, 424−428.(36) Na, Z.; Li, L.; Uttamchandani, M.; Yao, S. Q. Microarray-GuidedDiscovery of Two-Photon (2P) Small Molecule Probes for Live-CellImaging of Cysteinyl Cathepsin Activities. Chem. Commun. 2012, 48,7304−7306.(37) Li, L.; Zhang, C.-W.; Chen, G. Y. J.; Zhu, B.; Chai, C.; Xu, Q.-H.; Tan, E.-K.; Zhu, Q.; Lim, K. L.; Yao, S. Q. A Sensitive Two-PhotonProbe to Selectively Detect Monoamine Oxidase B Activity inParkinson’s Disease Models. Nat. Commun. 2014, 5, 3276.(38) Yan, S. Y.; Huang, R.; Wang, C.; Zhou, Y.; Wang, J.; Fu, B.;Weng, X.; Zhou, X. A Two-Photon Fluorescent Probe for IntracellularDetection of Tyrosinase Activity. Chem. - Asian J. 2012, 7, 2782−2785.(39) Li, L.; Ge, J.; Wu, H.; Xu, Q.-H.; Yao, S. Q. Organelle-SpecificDetection of Phosphatase Activities with Two-Photon FluorogenicProbes in Cells and Tissues. J. Am. Chem. Soc. 2012, 134, 12157−12167.(40) Kim, D.; Sambasivan, S.; Nam, H.; Kim, K. H.; Kim, J. Y.; Joo,T.; Lee, K. H.; Kim, K. T.; Ahn, K. H. Reaction-Based Two-PhotonProbes for in Vitro Analysis and Cellular Imaging of MonoamineOxidase Activity. Chem. Commun. 2012, 48, 6833−6835.(41) Frye, S. V. The Art of the Chemical Probe. Nat. Chem. Biol.2010, 6, 159−161.(42) Ostman, A.; Hellberg, C.; Bohmer, F. D. Protein-TyrosinePhosphatases and Cancer. Nat. Rev. Cancer 2006, 6, 307−320.(43) Gomori, G. Microtechnical Demonstration of Phosphatase inTissue Sections. Exp. Biol. Med. 1939, 42, 23−26.(44) Yudushkin, I. A.; Schleifenbaum, A.; Kinkhabwala, A.; Neel, B.G.; Schultz, C.; Bastiaens, P. I. H. Live-Cell Imaging of Enzyme-Substrate Interaction Reveals Spatial Regulation of PTP1B. Science2007, 315, 115−119.(45) Newman, R. H.; Zhang, J. Visualization of Phosphatase Activityin Living Cells with a FRET-Based Calcineurin Activity Sensor. Mol.BioSyst. 2008, 4, 496−501.(46) Sellars, J. D.; Landrum, M.; Congreve, A.; Dixon, D. P.; Mosely,J. A.; Beeby, A.; Edwards, R.; Steel, P. G. Fluorescence QuenchedQuinone Methide Based Activity Probes - a Cautionary Tale. Org.Biomol. Chem. 2010, 8, 1610−1618.(47) He, G. S.; Tan, L. S.; Zheng, Q.; Prasad, P. N. MultiphotonAbsorbing Materials: Molecular Designs, Characterizations, andApplications. Chem. Rev. 2008, 108, 1245−1330.(48) Mu, J.; Liu, F.; Rajab, M. S.; Shi, M.; Li, S.; Goh, C.; Lu, L.; Xu,Q.-H.; Liu, B.; Ng, L. G.; Xing, B. A Small-Molecule FRET Reporterfor the Real-Time Visualization of Cell-Surface Proteolytic EnzymeFunctions. Angew. Chem., Int. Ed. 2014, 53, 14357−14362.(49) Shih, J. C.; Chen, K.; Ridd, M. J. Monoamine Oxidase: fromGenes to Behavior. Annu. Rev. Neurosci. 1999, 22, 197−217.(50) Edmondson, D. E.; Mattevi, A.; Binda, C.; Li, M.; Hubalek, F.Structure and Mechanism of Monoamine Oxidase. Curr. Med. Chem.2004, 11, 1983−1993.(51) Tong, J.; Meyer, J. H.; Furukawa, Y.; Boileau, I.; Chang, L. J.;Wilson, A. A.; Houle, S.; Kish, S. J. Distribution of MonoamineOxidase Proteins in Human Brain: Implications for Brain ImagingStudies. J. Cereb. Blood Flow Metab. 2013, 33, 863−871.(52) Carradori, S.; Petzer, J. P. Novel Monoamine OxidaseInhibitors: a Patent Review (2012 – 2014). Expert Opin. Ther. Pat.2015, 25, 91−110.(53) Nicotra, A.; Pierucci, F.; Parvez, H.; Senatori, O. MonoamineOxidase Expression During Development and Aging. NeuroToxicology2004, 25, 155−165.(54) Kim, D.; Jun, Y. W.; Ahn, K. H. Fluorescent Probes for Analysisand Imaging of Monoamine Oxidase Activity. Bull. Korean Chem. Soc.2014, 35, 1269−1274.(55) Li, X.; Zhang, H.; Xie, Y.; Hu, Y.; Sun, H.; Zhu, Q. FluorescentProbes for Detecting Monoamine Oxidase Activity and Cell Imaging.Org. Biomol. Chem. 2014, 12, 2033−2036.(56) Li, X.; Yu, J.; Zhu, Q.; Qian, L.; Li, L.; Zheng, Y.; Yao, S. Q.Visualization of Monoamine Oxidases in Living Cells Using ″Turn-

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

633

Page 9: Two-Photon Small Molecule Enzymatic Probesiam.njtech.edu.cn/__local/8/BD/92/B1AAE7381233986A75AA...CONSPECTUS: Enzymes are essential for life, especially in the development of disease

ON″ Fluorescence Resonance Energy Transfer Probes. Analyst 2014,139, 6092−6095.(57) Long, S.; Chen, L.; Xiang, Y.; Song, M.; Zheng, Y.; Zhu, Q. AnActivity-Based Fluorogenic Probe for Sensitive and Selective Mono-amine Oxidase-B Detection. Chem. Commun. 2012, 48, 7164−7166.(58) Li, L.; Zhang, C.-W.; Ge, J.; Qian, L.; Chai, B.-H.; Zhu, Q.; Lee,J.-S.; Lim, K.-L.; Yao, S. Q. A Small-Molecule Probe for SelectiveProfiling and Imaging of Monoamine Oxidase B Activities in Modelsof Parkinson’s Disease. Angew. Chem., Int. Ed. 2015, 54, 10821−10825.(59) Turk, B. Targeting Proteases: Successes, Failures and FutureProspects. Nat. Rev. Drug Discovery 2006, 5, 785−799.(60) Vickers, C. J.; Gonzalez-Paez, G. E.; Wolan, D. W. SelectiveDetection and Inhibition of Active Caspase-3 in Cells with OptimizedPeptides. J. Am. Chem. Soc. 2013, 135, 12869−12876.(61) Jung, J. C.; Mehta, A. D.; Aksay, E.; Stepnoski, R.; Schnitzer, M.J. In Vivo Mammalian Brain Imaging Using One- and Two-PhotonFluorescence Microendoscopy. J. Neurophysiol. 2004, 92, 3121−3133.(62) Barretto, R. P. J.; Ko, T. H.; Jung, J. C.; Wang, T. J.; Capps, G.;Waters, A. C.; Ziv, Y.; Attardo, A.; Recht, L.; Schnitzer, M. J. Time-Lapse Imaging of Disease Progression in Deep Brain Areas UsingFluorescence Microendoscopy. Nat. Med. 2011, 17, 223−228.

Accounts of Chemical Research Article

DOI: 10.1021/acs.accounts.5b00512Acc. Chem. Res. 2016, 49, 626−634

634


Top Related